Recruitment of BAD by the Chlamydia trachomatis Vacuole Correlates with Host-Cell Survival.

Chlamydiae replicate intracellularly in a vacuole called an inclusion. Chlamydial-infected host cells are protected from mitochondrion-dependent apoptosis, partly due to degradation of BH3-only proteins. The host-cell adapter protein 14-3-3β can interact with host-cell apoptotic signaling pathways in a phosphorylation-dependent manner. In Chlamydia trachomatis-infected cells, 14-3-3β co-localizes to the inclusion via direct interaction with a C. trachomatis-encoded inclusion membrane protein. We therefore explored the possibility that the phosphatidylinositol-3 kinase (PI3K) pathway may contribute to resistance of infected cells to apoptosis. We found that inhibition of PI3K renders C. trachomatis-infected cells sensitive to staurosporine-induced apoptosis, which is accompanied by mitochondrial cytochrome c release. 14-3-3β does not associate with the Chlamydia pneumoniae inclusion, and inhibition of PI3K does not affect protection against apoptosis of C. pneumoniae-infected cells. In C. trachomatis-infected cells, the PI3K pathway activates AKT/protein kinase B, which leads to maintenance of the pro-apoptotic protein BAD in a phosphorylated state. Phosphorylated BAD is sequestered via 14-3-3β to the inclusion, but it is released when PI3K is inhibited. Depletion of AKT through short-interfering RNA reverses the resistance to apoptosis of C. trachomatis-infected cells. BAD phosphorylation is not maintained and it is not recruited to the inclusion of Chlamydia muridarum, which protects poorly against apoptosis. Thus, sequestration of BAD away from mitochondria provides C. trachomatis with a mechanism to protect the host cell from apoptosis via the interaction of a C. trachomatis-encoded inclusion protein with a host-cell phosphoserine-binding protein

Recruitment of BAD by the Chlamydia trachomatis vacuole correlates with host-cell survival

Chlamydiae replicate intracellularly in a vacuole called an inclusion. Chlamydial-infected host cells are protected from mitochondrion-dependent apoptosis, partly due to degradation of BH3-only proteins. The host-cell adapter protein 14-3-3β can interact with host-cell apoptotic signaling pathways in a phosphorylation-dependent manner. In Chlamydia trachomatis-infected cells, 14-3-3β co-localizes to the inclusion via direct interaction with a C. trachomatis-encoded inclusion membrane protein. We therefore explored the possibility that the phosphatidylinositol-3 kinase (PI3K) pathway may contribute to resistance of infected cells to apoptosis. We found that inhibition of PI3K renders C. trachomatis-infected cells sensitive to staurosporine-induced apoptosis, which is accompanied by mitochondrial cytochrome c release. 14-3-3β does not associate with the Chlamydia pneumoniae inclusion, and inhibition of PI3K does not affect protection against apoptosis of C. pneumoniae-infected cells. In C. trachomatis-infected cells, the PI3K pathway activates AKT/protein kinase B, which leads to maintenance of the pro-apoptotic protein BAD in a phosphorylated state. Phosphorylated BAD is sequestered via 14-3-3β to the inclusion, but it is released when PI3K is inhibited. Depletion of AKT through short-interfering RNA reverses the resistance to apoptosis of C. trachomatis-infected cells. BAD phosphorylation is not maintained and it is not recruited to the inclusion of Chlamydia muridarum, which protects poorly against apoptosis. Thus, sequestration of BAD away from mitochondria provides C. trachomatis with a mechanism to protect the host cell from apoptosis via the interaction of a C. trachomatis-encoded inclusion protein with a host-cell phosphoserine-binding protein

Genome-wide landscape of liver X receptor chromatin binding and gene regulation in human macrophages

<p>Abstract</p> <p>Background</p> <p>The liver X receptors (LXRs) are oxysterol sensing nuclear receptors with multiple effects on metabolism and immune cells. However, the complete genome-wide cistrome of LXR in cells of human origin has not yet been provided.</p> <p>Results</p> <p>We performed ChIP-seq in phorbol myristate acetate-differentiated THP-1 cells (macrophage-type) after stimulation with the potent synthetic LXR ligand T0901317 (T09). Microarray gene expression analysis was performed in the same cellular model. We identified 1357 genome-wide LXR locations (FDR < 1%), of which 526 were observed after T09 treatment. <it>De novo </it>analysis of LXR binding sequences identified a DR4-type element as the major motif. On mRNA level T09 up-regulated 1258 genes and repressed 455 genes. Our results show that LXR actions are focused on 112 genomic regions that contain up to 11 T09 target genes per region under the control of highly stringent LXR binding sites with individual constellations for each region. We could confirm that LXR controls lipid metabolism and transport and observed a strong association with apoptosis-related functions.</p> <p>Conclusions</p> <p>This first report on genome-wide binding of LXR in a human cell line provides new insights into the transcriptional network of LXR and its target genes with their link to physiological processes, such as apoptosis.</p> <p>The gene expression microarray and sequence data have been submitted collectively to the NCBI Gene Expression Omnibus <url>http://www.ncbi.nlm.nih.gov/geo</url> under accession number GSE28319.</p

Etude de la réponse inflammatoire au cours d'une infection par la bactérie intracellulaire stricte, chlamydia

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Effect of the purinergic receptor P2X7 on Chlamydia infection in cervical epithelial cells and vaginally-infected mice

Ligation of the purinergic receptor, P2X7R, with its agonist ATP has been previously shown to inhibit intracellular infection by chlamydiae and mycobacteria in macrophages. The effect of P2X7R on chlamydial infection had never been investigated in the preferred target cells of chlamydiae, cervical epithelial cells, nor in vaginally infected mice. In this study, we show that treatment of epithelial cells with P2X7R agonists inhibits partially Chlamydia infection in epithelial cells. Chelation of ATP with magnesium or pretreatment with a P2X7R antagonist blocks the inhibitory effects of ATP. Similarly to previous results obtained with macrophages, ATP-mediated inhibition of infection in epithelial cells requires activation of host-cell phospholipase D. Vaginal infection was also more efficient in P2X7R-deficient mice, which also displayed a higher level of acute inflammation in the endocervix, oviduct, and mesosalpingeal tissues than in infected wild-type mice. However, secretion of IL-1β, which requires P2X7R ligation during infection by other pathogens, was decreased mildly and only at short times of infection. Taken together, these results suggest that P2X7R affects Chlamydia infection by directly inhibiting infection in epithelial cells, rather than through the ability of P2X7R to modulate IL-1β secretion

Expression of purinergic receptors and modulation of P2X7 function by the inflammatory cytokine IFNγ in human epithelial cells

AbstractThe cervical epithelial cell line, HeLa, is one of the oldest and most commonly used cell lines in cell biology laboratories. Although a truncated P2X7 receptor has recently been identified in HeLa cells, the expression of other purinergic receptors or the function of the P2X7 protein has not been characterized. We here show that HeLa cells express transcripts for most P2X and P2Y purinergic receptors. Treatment of cells with ATP or other P2X7 agonists does not stimulate cell death, but can induce atypical calcium fluxes and ion currents. Cervical epithelial cells represent an important target for sexually-transmitted pathogens and are commonly exposed to pro-inflammatory cytokines such as IFNγ. Stimulation of HeLa cells with IFNγ upregulates expression of P2X7 mRNA and full-length protein, modifies ATP-dependent calcium fluxes, and renders the cells sensitive to ATP-induced apoptosis, which can be blocked by a P2X7 antagonist. IFNγ treatment also increased dramatically the sensitivity of the intestinal epithelial cell line, HCT8, to ATP-induced apoptosis. Significantly, IFNγ also stimulated P2X7 expression on human intestinal tissues. Responses to other purinergic receptor ligands suggest that HeLa cells may also express functional P2Y1, P2Y2 and P2Y6 receptors, which could be relevant for modulating ion homeostasis in the cells

AKT Phosphorylation in Cells during Apoptosis of C. trachomatis-Infected Cells

<p>Cells were infected with C. trachomatis at an MOI of 1.0 for 24 or 48 h, or mock-infected, and then incubated with 50 μM LY294002 (LY) or control buffer for 6 h before treating with control buffer or 2 μM STS overnight. Cells were then collected for Western immunoblotting and analyzed for phosphorylation of AKT on residue Ser⁴⁷³, as described in Materials and Methods. STS treatment by itself did not affect AKT Ser⁴⁷³ phosphorylation in infected cells, but LY294002 caused AKT Ser⁴⁷³ to become partially dephosphorylated. This residue became dephosphorylated almost completely when LY294002 was combined with STS in infected cells. One experiment of two representative experiments performed on separate days is shown.</p

Effect of PI3K on BAD Phosphorylation during C. trachomatis Infection

<p>Cells over-expressing BAD were infected with C. trachomatis at an MOI of 1.0 for 26 h or mock-infected, and then incubated with 50 μM LY294002 (LY) or control buffer for 6 h before treating with 2 μM STS overnight. Cells were then collected for Western immunoblotting and analyzed for total BAD protein (top row), phosphorylation of BAD on residue Ser¹³⁶ (middle row), or total P42 MAP kinase protein (bottom row), as described in Materials and Methods. C. trachomatis infection led to a decrease in the level of BAD, which was further decreased by STS treatment. Pre-incubation with LY294002 did not further alter the level of total BAD (top row). STS-treatment of uninfected cells caused complete dephosphorylation of BAD, but BAD remained phosphorylated after STS-treatment of infected cells (middle row). However, pre-incubation of infected cells with LY294002 before treatment with STS resulted in complete dephosphorylation of BAD (middle row). Infection had no effect on total P42 MAP kinase protein levels (bottom row). One experiment of three representative experiments performed on separate days is shown.</p